Project summary Neural circuits are groups of neurons that respond to stimuli and signal to each other to form functional units of the brain. When compromised, they are thought to cause brain disorders such as anxiety and depression. However, it remains difficult to understand how abnormalities in neural circuits arise because we have yet to understand how all the cells in any neural circuit interact normally. Due to its simple anatomy and easily-read output, the C. elegans egg-laying circuit is intensively studied as a model for neural circuits. Even so, the egg- laying circuit shows phenomena that cannot be explained, such as coordination between egg-laying and locomotion behaviors, that suggest the circuit may have components that currently remain unknown. I have discovered what may be a previously unrecognized neuron of the egg-laying system, and here I describe my plans to utilize the many advantages this model circuit to characterize the functional role of this neuron in the circuit. I initially discovered new neural structures lying immediately over the egg-laying system by examining multiple transgenic strains that express fluorescent proteins in various subsets of C. elegans neurons. After creating a GFP reporter transgene that labels only the cells that create these structures, I successfully identified the structures to be branches off of the main processes of the two PVP neurons, whose functions have not yet been determined. I used confocal microscopy to find that (a) the branches terminate in winged- shaped sensory cilia; (b) the cilia develop concurrently as the rest of the egg-laying circuit differentiates; (c) the PVP cilia are specific to hermaphrodites, and are not present in males (which lack an egg-laying circuit); and (d) the PVP cilium vary in morphology and position from animal to animal. Furthermore, by expressing a histamine-gated chloride channel specifically in PVP, I inhibited PVP activity in adult animals and observed resulting changes in locomotion behavior, including increased reversals and decreased forward speed. This, confirms a functional role for PVPs in controlling locomotion that had been hinted at by earlier studies. My project tests the hypothesis that PVP uses its cilium to sense function of the egg-laying circuit and coordinates locomotion with egg laying. To investigate this hypothesis, in Aim 1 I will visualize PVP in conjunction with markers of other structures in the egg-laying anatomy to better understand the PVP cilium's position and morphology in relation to the egg-laying circuit. In Aim 2, I will inhibit PVP activity and determine if and how PVP functionally affects egg-laying and locomotion behaviors using a battery of behavioral assays. Lastly, in Aim 3 I will express a calcium-sensitive fluorescent reporter in PVP and observe when PVP is active in freely- behaving animals and moreover, using PVP-specific RNAi to disrupt the structure and function of PVP cilia, determine if the PVP cilia are necessary for PVP cell activity and function. Together, these aims will characterize a missing link in what was already one of the best-characterized neural circuits in any organism, advancing our understanding of how neural circuits work.